Signaling-mediated bacterial persister formation

Abstract

Here we show that bacterial communication through indole signaling induces persistence, a phenomenon in which a subset of an isogenic bacterial population tolerates antibiotic treatment. We monitor indole-induced persister formation using microfluidics and identify the role of oxidative-stress and phage-shock pathways in this phenomenon. We propose a model in which indole signaling 'inoculates' a bacterial subpopulation against antibiotics by activating stress responses, leading to persister formation.

Figure 1. Indole induces persistence in E. coli

All experiments were performed at 37°C. (a) Percent survival of stationary-phase wild-type, ΔtnaA, and Δmtr E. coli in M9CG and in rich media (LB), pre-incubated with and without indole and treated with ofloxacin. Error bars represent mean ± s.d. of at least three biological replicates. (b) Direct observation of indole-induced persistence. Representative time-lapse of optical (DIC) and fluorescence (GFP) images of wild-type E. coli PtnaC grown in the microfluidic chamber in selective media (M9CG + kanamycin) for two hours (I), then treated with 500 μM indole (1 hour, II) before lysis with ampicillin (30 minutes, III; 1 hour, IV; 1 hour ampicillin lysis + 42 minutes in selective media, V). Time (in hours:minutes) depicted within each image corresponds to total time elapsed since the beginning of the experiment. Raw fluorescent images were identically exposed and contrast-scaled. Scale bar is 5 μM. (c) Histograms of normalized PtnaC activity (see Supplementary Methods), for lysed versus surviving cells in microfluidics experiments. Data were obtained from three biological replicates.

Figure 2. Indole induces persistence through the phage shock and OxyR pathways

All experiments were performed in M9CG. Error bars represent mean ± s.d. of at least three biological replicates. (a) Fold change survival in indole-treated versus untreated stationary phase cultures of wild-type E. coli, ΔfluΔoxyR, and ΔpspBC after treatment with ofloxacin. (b) Pre-treatment of stationary phase cultures of wild-type E. coli with hydrogen peroxide leads to increased survival after subsequent ofloxacin treatment. Black line indicates percent survival of cultures after incubation for 1 hour with hydrogen peroxide. Grey bars indicate percent survival of the same cultures after subsequent ofloxacin treatment, relative to survival after incubation with H₂O₂. (c) Proposed mechanism for indole-induced persister formation. Up-regulated gene products and pathways are shown in red and down-regulated gene products and pathways are shown in blue (see Supplementary Fig. 16 and 17).